Full (127 KB) - Bahrain Medical Bulletin

Bahrain Medical Bulletin, Vol. 34, No. 1, March 2012
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Detection of Multidrug-Resistant Tuberculosis Using PCR Compared to the Conventional
Proportional Method
Mogahid M Elhassan, PhD* Samar M Saeed, Msc** Miskelyemen A Elmekki, PhD***
Ahmed A Al-Jarie, MD**** Mohamed E Hamid, PhD*****
Objective: To evaluate the PCR technique for the rapid defection of Multidrug-Resistant (MDR)
Mycobacterium tuberculosis compared to the conventional proportional drug sensitivity testing.
Design: Cross sectional laboratory based study.
Setting: Alshaab Teaching Hospital, Abu-Angah Hospital and the National Health Laboratory,
Sudan.
Method: One hundred thirty tuberculosis suspected individuals of both sexes and of different
ages were included in the study. Sputum samples were cultured on Lowenstein-Jensen (LJ)
medium. Resistant strains were tested for the presence of mutations conferring resistance using
molecular techniques to amplify 315 base pair (bp) rifampicin (RIF) and 146 bp isoniazid (INH),
as markers for MDR among Mycobacterium tuberculosis.
Result: One hundred nineteen (91.5%) showed Mycobacterium tuberculosis-like colonies, 65 of
which were randomly subjected to PCR and examined for the presence of IS6110 insertion
sequences. Fifty-six (86.2%) were confirmed members of the Mycobacterium tuberculosis. The
result of antibiotics susceptibility testing revealed that 32/56 (57.1%) of the strains were resistant
to RIF, 36/56 (64.3%) to INH and 30/56 (53.6%) were resistant to both drugs (MDR). The
conventional method showed 21/56 (37.5%) were resistant to RIF, 32/56 (57.1%) to INH and
16/56 (28.6%) were resistant to both drugs (MDR).
Conclusion: Not all resistant strains detected by conventional were detected by PCR method; 14
(25%) were missed for RIF, 9 (16.1%) for INH and 4 (7.1%) for both. This represents a
significant lack of sensitivity of the PCR technique, which could be due to the presence of other
types of mutations that needs other primers and PCR protocol.
Bahrain Med Bull 2012; 34(1):
____________________________________________________________________________
* Associate Professor and Head
Department of Microbiology
** Lecture and Researcher
Department of Microbiology
Sudan University of Science and Technology
*** Assistant Professor of Immunology and Head Department of Parasitology
Sudan University of Science and Technology
**** Consultant of Pediatrics Infectious Diseases and Dean College of Al-Ghad International
Medical Science Colleges, KSA
***** Associate Professor and Consultant of Microbiology
Tuberculosis Research Group, Department of Microbiology
King Khalid University, KSA
Email: mogahidelhassan@yahoo.com
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The most effective anti-TB drugs are isoniazid (INH) and rifampicin (RIF). Resistant Mycobacteria to
at least one of these drugs are the cause of Multidrug-resistant tuberculosis (MDR-TB). This type of
resistance is highly problematic due to limited sources of drugs as well as the high toxicity, low
efficacy and high cost of second-line tuberculosis drugs 1 .
In 2008, an estimated 390,000 - 510,000 cases of MDR-TB emerged globally. Among TB cases, 3.6%
are estimated to have MDR-TB. Twenty-two of 48 African countries reported first-line TB drug
resistance, the estimated number of MDR-TB cases (primary and acquired) in 2008 was 69,000
(53,000 - 110,000) 2 .
Since the early study by Cavanagh, data on drug resistant TB are lacking in the Sudan 3 . Tuberculosis
control program in Khartoum 2006 showed that there was no system to detect the prevalence of MDR-
TB or HIV among the TB cases 4 .
Although the rate of drug resistance is continuously increasing, only around 7% of estimated cases are
detected. The control of drug resistant disease is difficult especially in high burden countries due to
poor laboratory services and the slow nature of conventional drug susceptibility testing 5 .
Therefore, implementation of rapid molecular methods for detecting drug-resistant TB may be a viable
alternative to culture-based DST. Recently the WHO recommended the use of molecular techniques
such as Line probe assay (LPA) for rapid screening of MDR-TB in low and middle-income settings 6 .
The role of mycobacterial catalase-peroxidase gene (katG) was determined by cloning and sequencing
of this gene. Mutations in this gene were found in 42 - 58% of isoniazid-resistant clinical isolates,
confirming the effect of KatG enzyme in INH activity 7 .
Other mutations were detected in Ser315Thr, about 40% of isoniazid-resistant strains 8 . This mutation
was found to result in the production of a catalase enzyme that retains about 50% of isoniazid catalaseperoxidase
activity, which is sufficient for the ability of the organism to evade the action of host active
radicals 9 .
Spontaneous mutations (deletions/substitutions/insertions) were found to occur in the 81-bp hotspot
region of the rpoβ gene which encodes DNA-dependent RNA polymerase (the target for rifampicin
binding); this result into replacement of the aromatic with non-aromatic amino acids in the target RNA
polymerase enzyme, which consequently leads to poor bonding between rifampicin and the RNA
polymerase 10,11 .
The aim of this study is to evaluate the PCR technique for the rapid defection of MDR Mycobacterium
tuberculosis in comparison to the conventional proportional drug sensitivity testing.
METHOD
Randomized prospective cross sectional study was performed. Smear-positive sputum was collected
from 130 patients with persistent tuberculosis. Informed consent was obtained from each patient. The
study was approved by the Federal Ministry of Health and the ethical committee of the Scientific
Research Council of Sudan University of Science and Technology.
Sputum samples were screened for acid-fast bacilli (AFBs) using Ziehl-Neelsen (ZN) smear
microscopy. Handling and processing of specimens were performed in biosafety cabinet (BSL 3).
Sputum was processed with the N-acetyl-L-cysteine-sodium hydroxide (NaOH) method (NaOH final
concentration, 1.5%) and cultured in Lowenstein-Jensen (LJ) slants. Isolation and conventional
identification of Mycobacterium tuberculosis were performed following standard method 12 . Drug
susceptibility testing was performed by LJ proportion method 13 .
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Sixty-five Mycobacterium tuberculosis isolates were randomly selected from the positive cultures and
were confirmed as Mycobacterium tuberculosis by detecting species-specific IS6110 (123 bp) using
primers and method previously described 14 . Resistant strains detected by conventional methods were
tested for the presence of mutations conferring resistance to isoniazid (INH) and rifampicin (RIF)
using PCR method.
SPSS for windows version 11.0 was used for data analysis.
RESULT
One hundred thirty sputum specimens from patients were examined, 82 males and 48 females, age
range was 12 to 67 years, 119 (91.5%) showed Mycobacterium tuberculosis-like colonies, 65 were
randomly subjected to PCR and examined for the presence of IS6110 insertion sequences. PCR
revealed that 56 (86.2%) were Mycobacterium tuberculosis and 9 (13.8%) were MOTTs, see table 1
and figure 1.
Table 1: Susceptibility Testing of IS6110 Positive Mycobacterium Tuberculosis for Rifampicin
and Isoniazid by Proportion Method Compared to Multiplex PCR Method
LJ proportion method PCR method
RIF INH
Number (%)
RIF INH
Number (%)
Resistant 32 (57.1%) 36 (64.3%) 21 (37.5%) 32 (57.1%)
Sensitive 23 (41.1%) 19 (33.9%) 35 (62.5%) 24 (42.9%)
ND 1 (1.8%) 1 (1.8%) - -
Total 56 56 56 56
Figure 1: PCR Amplified IS6110 Sequences, Lane 1, DNA Marker; Lane 2, Negative Control;
Lane 3, Reference Mycobacterium Tuberculosis Strain H37v (Positive Control)
Antibiotics susceptibility testing revealed that 32/56 (57.1%) of the strains were resistant to RIF using
the conventional proportion method compared to 21/56 (37.5%) using PCR method. Resistant for INH
were 36/56 (64.3%) using the conventional method compared to 32/56 (57.1%) using PCR method;
51.8% of the strains were found resistant for both drugs (MDR) using the conventional method
compared to 28.6% for both drugs (MDR) using the PCR method, see figures 2 and 3.
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Figure 2: The Amplicon of Rifampicin Resistant Mycobacterium Tuberculosis; Lane 1, DNA
Marker; Lane 2, Negative Control; Lane 3, Positive Control; Lane 7; RIF Resistant Strain
Figure 3: The Amplicon of Isoniazid Resistant Mycobacterium Tuberculosis; Lane 1, DNA
Marker; Lane 2, Negative Control; Lane 3, Positive Control; Lanes 4 And 7, INH Resistant
Strains
DISCUSSION
The result of antibiotics susceptibility testing revealed that there were obvious differences between the
conventional and PCR method, this may be due to the presence of resistant mutations, which could not
be detected using current primers.
It was noticed that the majority of the isolates, which showed resistance to the two drugs on LJ
medium proportion method showed resistance on the PCR to the two drugs or at least to one of them,
mostly isoniazid; therefore, resistance to Isoniazid can be considered as a marker for multidrug
resistance 15 . However, Sharma et al considered resistance to rifampicin as a marker for MDR TB 16 .
In this study, a rising resistance rates of MDR among TB patients in Sudan compared with some
African countries were revealed. In Ethiopia 14% of the isolates were found MDR, in Kenya 33.3%, in
South Africa 60.2% and in Saudi Arabia 11% resistance to isoniazid and 9.7% to rifampicin was
recorded 17-20 . In the present study, a rate of 21.5% was detected, which could be compared to Sharaf el
Din et al who applied PCR based dot-blot method and found that 12% of strains were resistant to INH
(katG gene), 8% were resistant to RIF (rpoB gene), 30% to STM (rpsl gene) and 4% to EMB (embB
gene) but no record of MDR was investigated by these authors in patients with persistent TB 21 .
Similarly, a novel PCR-based reverse hybridization method Genotype MTBDR assay (Hain
Lifescience GmbH, Nehren, Germany) was evaluated for rapid detection of rifampicin (RIF) and
isoniazid (INH) resistance in Turkish Mycobacterium tuberculosis isolates revealed that RIF resistance
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was correctly identified in 95.1% of the isolates and in 73% of INH-resistant isolates. However, the
recommendation of the author is to confirm the result with phenotypic methods 22 .
The GenoType® MTBDRplus assay has been validated as a rapid and reliable first-line diagnostic test
on AFB-positive sputum or MTB isolates for INH resistance, RIF resistance and MDR-TB in
Bangkok, it was found reliable, but its impact on treatment outcome, feasibility and the cost associated
with widespread implementation needs to be evaluated 23 .
The presence of some isolates in this study, which are sensitive in the proportional method and
appeared as resistant in the PCR to one of the two drugs or both of them could not be attributed to
contamination, rather it could be due to mutations, which are not necessarily strongly related to
resistance. Moreover, since the target region of the genes for RIF and INH resistance are not
mentioned by the manufacturer, it is difficult to explain this part of the result.
CONCLUSION
Classical DST is still a gold standard method for diagnosis of MDR TB in developing countries.
However, substitution of the classical DST with the rapid and sensitive PCR needs to be
carefully evaluated as the protocol used in this study failed to detect all types of mutations
present. Other PCR protocols targeting all known mutations in RIF and INH genes could be of
more useful.
___________________________________________________________________________
Author contribution: All authors share equal effort contribution towards (1) substantial contributions
to conception and design, acquisition, analysis and interpretation of data; (2) drafting the article and
revising it critically for important intellectual content; and (3) final approval of the manuscript version
to be published. Yes
Potential conflicts of interest: No
Competing interest: None Sponsorship: None
Submission date: 30 March 2011 Acceptance date: 2 January 2012
Ethical approval: The study was approved by the ethical committee of Federal Ministry of Health
and the ethical committee of research council of Sudan University of Science and Technology.
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